ABSTRACT Aminoacyl-tRNA synthetases (aaRSs) are enzymes that activate L-amino acids for protein synthesis. However, during evolution, aaRSs progressively accrued ?moonlighting? functions that are activated under conditions of diminished protein synthesis and that enable aaRSs to regulate metabolic homeostasis and modulate signal transduction pathways. In particular, Tyrosyl-tRNA synthetase (TyrRS) moves into the nucleus under stress conditions and facilitates DNA repair through poly-ADP-ribose polymerase 1 (PARP1) activation. PARP1, the central regulator of nicotinamide adenine dinucleotide (NAD+) signaling, senses and responds to DNA damage and activates DNA repair pathways. Unrepaired or erroneously repaired DNA double strand breaks (DSBs) in neurons are a major contributing factor in the development of a variety of neurological disorders including Alzheimer?s disease (AD). In particular, accumulation of amyloid ??(A?)? a hallmark of AD, exacerbates the accumulation of DSBs in neurons. Remarkably, PARP1 is downregulated in AD and the TyrRS-PARP1 pathway was shown to attenuate A?-induced neurotoxicity. Stimulation of this pathway could offer a new approach to the treatment of AD and other neurodegenerative diseases. We hypothesize that the L-tyrosine binding pocket of eukaryotic TyrRS can be targeted to design and develop small molecules named TyrRS- Targeting Compounds (TTCs), which will activate the moonlighting functions of TyrRS that stimulate DNA repair, and that such molecules will exert neuroprotective activities in AD-relevant models. In the first year of this project, we have designed and synthesized a series of TTCs on the basis of our work published in Nature, which demonstrated that the cis-isomer of resveratrol (RSV) binds to TyrRS and acts as an L-tyrosine antagonist, activating PARP1-dependent signaling cascades. Some of the newly synthesized TTCs have already shown neuroprotective activity in vitro. Mechanistic analysis indicated that the lead neuroprotective compound activates AKT and upregulates DNA repair proteins. In the remaining period, we will pursue the following Specific Aims. Under Aim 1, we will continue structure-based design and synthesis of TyrRS Targeting Compounds (TTCs) based on the cis-RSV-bound co-crystal structure of TyrRS and demonstrate that such molecules engage and affect their TyrRS target. Under Aim 2, we will determine the effect of TTCs on the survival of primary cortical neurons and TyrRS/PARP1-dependent DNA repair and signaling cascades. Under Aim 3, we will test the in vivo therapeutic potential of TTCs that show neuroprotection in vitro using 5XFAD mouse model of AD. The completion of these Aims will provide proof-of-principle data that would guide the eventual development of a new class of drugs that targets TyrRS to activate PARP1-dependent DNA repair in neurons, with potential applicability to AD and other neurodegenerative diseases that originate from accumulated DNA damage.